Purification and mechanistic characterisation of two polygalacturonases from Sclerotium rolfsii

Sclerotium rolfsii (strain CBS 350.80) was found to produce extraordinary high amounts of polygalacturonases (PGs). Two of these extracellular enzymes were purified by a recently introduced preparative electrophoretic device (isoelectric focusing mode of free flow electrophoresis). PG 1 (39.5 kDa, pI 6.5) and PG 2 (38 kDa, pI 5.4) exhibited quite similar properties, they were found to be both endo-acting enzymes. Both PGs cleaved penta- and trigalacturonic acid while tetragalacturonic acid was only cleaved when trigalacturonic acid was present. The latter substrate was hydrolysed much faster by PG 2. Both enzymes were active on pectins with different degrees of esterification, they were sensitive towards Ca-cations and not glycosylated. The kinetic properties were measured by viscosimetry with polygalacturonic acid as a substrate. NMR experiments on a model substrate revealed an inverting mechanism of carbohydrate hydrolysis for both enzymes

Missing peroxy radical sources within a summertime ponderosa pine forest

Organic peroxy (RO₂) and hydroperoxy (HO₂) radicals are key intermediates in the photochemical processes that generate ozone, secondary organic aerosol and reactive nitrogen reservoirs throughout the troposphere. In regions with ample biogenic hydrocarbons, the richness and complexity of peroxy radical chemistry presents a significant challenge to current-generation models, especially given the scarcity of measurements in such environments. We present peroxy radical observations acquired within a ponderosa pine forest during the summer 2010 Bio-hydro-atmosphere interactions of Energy, Aerosols, Carbon, H₂O, Organics and Nitrogen – Rocky Mountain Organic Carbon Study (BEACHON-ROCS). Total peroxy radical mixing ratios reach as high as 180 pptv (parts per trillion by volume) and are among the highest yet recorded. Using the comprehensive measurement suite to constrain a near-explicit 0-D box model, we investigate the sources, sinks and distribution of peroxy radicals below the forest canopy. The base chemical mechanism underestimates total peroxy radicals by as much as a factor of 3. Since primary reaction partners for peroxy radicals are either measured (NO) or underpredicted (HO₂ and RO₂, i.e., self-reaction), missing sources are the most likely explanation for this result. A close comparison of model output with observations reveals at least two distinct source signatures. The first missing source, characterized by a sharp midday maximum and a strong dependence on solar radiation, is consistent with photolytic production of HO₂. The diel profile of the second missing source peaks in the afternoon and suggests a process that generates RO₂ independently of sun-driven photochemistry, such as ozonolysis of reactive hydrocarbons. The maximum magnitudes of these missing sources (~120 and 50 pptv min⁻¹, respectively) are consistent with previous observations alluding to unexpectedly intense oxidation within forests. We conclude that a similar mechanism may underlie many such observations

Missing Peroxy Radical Sources Within a Rural Forest Canopy

Organic peroxy (RO2) and hydroperoxy (HO2) radicals are key intermediates in the photochemical processes that generate ozone, secondary organic aerosol and reactive nitrogen reservoirs throughout the troposphere. In regions with ample biogenic hydrocarbons, the richness and complexity of peroxy radical chemistry presents a significant challenge to current-generation models, especially given the scarcity of measurements in such environments. We present peroxy radical observations acquired within a Ponderosa pine forest during the summer 2010 Bio-hydro-atmosphere interactions of Energy, Aerosols, Carbon, H2O, Organics and Nitrogen - Rocky Mountain Organic Carbon Study (BEACHON-ROCS). Total peroxy radical mixing ratios reach as high as 180 pptv and are among the highest yet recorded. Using the comprehensive measurement suite to constrain a near-explicit 0-D box model, we investigate the sources, sinks and distribution of peroxy radicals below the forest canopy. The base chemical mechanism underestimates total peroxy radicals by as much as a factor of 3. Since primary reaction partners for peroxy radicals are either measured (NO) or under-predicted (HO2 and RO2, i.e. self-reaction), missing sources are the most likely explanation for this result. A close comparison of model output with observations reveals at least two distinct source signatures. The first missing source, characterized by a sharp midday maximum and a strong dependence on solar radiation, is consistent with photolytic production of HO2. The diel profile of the second missing source peaks in the afternoon and suggests a process that generates RO2 independently of sun-driven photochemistry, such as ozonolysis of reactive hydrocarbons. The maximum magnitudes of these missing sources (approximately 120 and 50 pptv min1, respectively) are consistent with previous observations alluding to unexpectedly intense oxidation within forests. We conclude that a similar mechanism may underlie many such observations

An acid-stable laccase from sclerotium rolfsii with potential for wool dye decolourization

The plant pathogen basidiomycete S. rolfsii secretes two laccases (SRL1 and SRL2) with molecular weights of 55 and 86 kDa, respectively. Laccase production was shown to be inducible by the addition of 2,5-xylidine to the cultural media. After treatment with a combination of chitinase and -1,3-glucanase, two different laccases were isolated from the sclerotia depending on the stage of sclerotia development. The more prominent laccase, SRL1, was purified and found to decolourize the industrially important wool azo dye Diamond Black PV 200 without the addition of redox mediators. The enzyme (pI 5.2) was active in the acidic pH range, showing an optimal activity at pH 2.4, with ABTS as substrate. The optimum temperature for activity was determined to be 62 ◦C. Enzyme stability studies revealed that SRL1 was notably stable at 18 ◦C and pH 4.5, retaining almost full activity after a week. Oxidation of tyrosine was not detectable under the reaction conditions but the enzyme did oxidize a variety of the usual laccase substrates. SRL1 was strongly inhibited by sodium azide and fluoride. Dye solutions decolourized with the immobilized laccase were successfully used for redyeing.(undefined

Comparison of different real time VOC measurement techniques in a ponderosa pine forest

Volatile organic compound (VOC) mixing ratios measured by five independent instruments are compared at a forested site dominated by ponderosa pine (Pinus Ponderosa) during the BEACHON-ROCS field study in summer 2010. The instruments included a Proton Transfer Reaction Time of Flight Mass Spectrometer (PTR-TOF-MS), a Proton Transfer Reaction Quadrupole Mass Spectrometer (PTR-MS), a Fast Online Gas-Chromatograph coupled to a Mass Spectrometer (GC/MS; TOGA), a Thermal Dissociation Chemical Ionization Mass Spectrometer (PAN-CIMS) and a Fiber Laser-Induced Fluorescence Instrument (FILIF). The species discussed in this comparison include the most important biogenic VOCs and a selected suite of oxygenated VOCs that are thought to dominate the VOC reactivity at this particular site as well as typical anthropogenic VOCs that showed low mixing ratios at this site. Good agreement was observed for methanol, the sum of the oxygenated hemiterpene 2-methyl-3-buten-2-ol (MBO) and the hemiterpene isoprene, acetaldehyde, the sum of acetone and propanal, benzene and the sum of methyl ethyl ketone (MEK) and butanal. Measurements of the above VOCs conducted by different instruments agree within 20%. The ability to differentiate the presence of toluene and cymene by PTR-TOF-MS is tested based on a comparison with GC-MS measurements, suggesting a study-average relative contribution of 74% for toluene and 26% for cymene. Similarly, 2-hydroxy-2-methylpropanal (HMPR) is found to interfere with the sum of methyl vinyl ketone and methacrolein (MVK + MAC) using PTR-(TOF)-MS at this site. A study-average relative contribution of 85% for MVK + MAC and 15% for HMPR was determined. The sum of monoterpenes measured by PTR-MS and PTR-TOF-MS was generally 20–25% higher than the sum of speciated monoterpenes measured by TOGA, which included α-pinene, β-pinene, camphene, carene, myrcene, limonene, cineole as well as other terpenes. However, this difference is consistent throughout the study, and likely points to an offset in calibration, rather than a difference in the ability to measure the sum of terpenes. The contribution of isoprene relative to MBO inferred from PTR-MS and PTR-TOF-MS was smaller than 12% while GC-MS data suggested an average of 21% of isoprene relative to MBO. This comparison demonstrates that the current capability of VOC measurements to account for OH reactivity associated with the measured VOCs is within 20%

Comparison of different real time VOC measurement techniques in a ponderosa pine forest

Volatile organic compound (VOC) mixing ratios measured by five independent instruments are compared at a forested site dominated by ponderosa pine (Pinus Ponderosa) during the BEACHON-ROCS field study in summer 2010. The instruments included a Proton Transfer Reaction Time of Flight Mass Spectrometer (PTR-TOF-MS), a Proton Transfer Reaction Quadrupole Mass Spectrometer (PTR-MS), a Fast Online Gas-Chromatograph coupled to a Mass Spectrometer (GC/MS; TOGA), a Thermal Dissociation Chemical Ionization Mass Spectrometer (PAN-CIMS) and a Fiber Laser-Induced Fluorescence Instrument (FILIF). The species discussed in this comparison include the most important biogenic VOCs and a selected suite of oxygenated VOCs that are thought to dominate the VOC reactivity at this particular site as well as typical anthropogenic VOCs that showed low mixing ratios at this site. Good agreement was observed for methanol, the sum of the oxygenated hemiterpene 2-methyl-3-buten-2-ol (MBO) and the hemiterpene isoprene, acetaldehyde, the sum of acetone and propanal, benzene and the sum of methyl ethyl ketone (MEK) and butanal. Measurements of the above VOCs conducted by different instruments agree within 20%. The ability to differentiate the presence of toluene and cymene by PTR-TOF-MS is tested based on a comparison with GC-MS measurements, suggesting a study-average relative contribution of 74% for toluene and 26% for cymene. Similarly, 2-hydroxy-2-methylpropanal (HMPR) is found to interfere with the sum of methyl vinyl ketone and methacrolein (MVK + MAC) using PTR-(TOF)-MS at this site. A study-average relative contribution of 85% for MVK + MAC and 15% for HMPR was determined. The sum of monoterpenes measured by PTR-MS and PTR-TOF-MS was generally 20–25% higher than the sum of speciated monoterpenes measured by TOGA, which included α-pinene, β-pinene, camphene, carene, myrcene, limonene, cineole as well as other terpenes. However, this difference is consistent throughout the study, and likely points to an offset in calibration, rather than a difference in the ability to measure the sum of terpenes. The contribution of isoprene relative to MBO inferred from PTR-MS and PTR-TOF-MS was smaller than 12% while GC-MS data suggested an average of 21% of isoprene relative to MBO. This comparison demonstrates that the current capability of VOC measurements to account for OH reactivity associated with the measured VOCs is within 20%

Evaluation of HO<sub>x</sub> sources and cycling using measurement-constrained model calculations in a 2-methyl-3-butene-2-ol (MBO) and monoterpene (MT) dominated ecosystem

We present a detailed analysis of OH observations from the BEACHON (Bio-hydro-atmosphere interactions of Energy, Aerosols, Carbon, H2O, Organics and Nitrogen)-ROCS (Rocky Mountain Organic Carbon Study) 2010 field campaign at the Manitou Forest Observatory (MFO), which is a 2-methyl-3-butene-2-ol (MBO) and monoterpene (MT) dominated forest environment. A comprehensive suite of measurements was used to constrain primary production of OH via ozone photolysis, OH recycling from HO2, and OH chemical loss rates, in order to estimate the steady-state concentration of OH. In addition, the University of Washington Chemical Model (UWCM) was used to evaluate the performance of a near-explicit chemical mechanism. The diurnal cycle in OH from the steady-state calculations is in good agreement with measurement. A comparison between the photolytic production rates and the recycling rates from the HO2 + NO reaction shows that recycling rates are ~20 times faster than the photolytic OH production rates from ozone. Thus, we find that direct measurement of the recycling rates and the OH loss rates can provide accurate predictions of OH concentrations. More importantly, we also conclude that a conventional OH recycling pathway (HO2 + NO) can explain the observed OH levels in this non-isoprene environment. This is in contrast to observations in isoprene-dominated regions, where investigators have observed significant underestimation of OH and have speculated that unknown sources of OH are responsible. The highly-constrained UWCM calculation under-predicts observed HO2 by as much as a factor of 8. As HO2 maintains oxidation capacity by recycling to OH, UWCM underestimates observed OH by as much as a factor of 4. When the UWCM calculation is constrained by measured HO2, model calculated OH is in better agreement with the observed OH levels. Conversely, constraining the model to observed OH only slightly reduces the model-measurement HO2 discrepancy, implying unknown HO2 sources. These findings demonstrate the importance of constraining the inputs to, and recycling within, the ROx radical pool (OH + HO2 + RO2)